PROJECT SUMMARY ? PROJECT 3 (ROCKEFELLER AND MOUNT SINAI) In addition to post-translational modifications of histones and DNA, and the 3D organization of chromatin, eukaryotic cells further generate chromatin structural variation by introducing variant histone proteins into existing or new nucleosomes. Over the past four years, Project 3 established that H3.3, a variant of histone H3, is uniquely incorporated into neuronal chromatin during development in an activity-dependent manner. Moreover, any ongoing turnover of nucleosome assembly requires H3.3, and such dynamic regulation is required both for cell type-specific specification of gene expression during development as well as for the transcriptional and synaptic plasticity that occurs in neurons in several limbic brain regions as a function of behavioral experience. More recently, we found that H3.3 is induced selectively in nucleus accumbens (NAc) by chronic stress in adult mice and by depression in humans, effects seen in males and females. Moreover, exposure to early life stress triggers a gradually accumulating skew in H3.3 expression in NAc, such that by adulthood there is a large increase in H3.3:H3.1/2 ratios in this brain region of mice exposed to stress early in life. Finally, selective knockdown of H3.3 in adult NAc neurons exerts a pro-resilience effect. We will now define whether H3.3 regulation controls depression-related phenomena at the level of D1- vs. D2-type medium spiny neurons (MSNs) in NAc and use this regulation as a novel means of identifying the genomic loci where nucleosome turnover is pathologically affected in depression. We will thus employ ATAC-seq and RNA-seq, with the Chromatin and Gene Analysis Core, to map nucleosome positioning and gene expression genome- wide in D1 and D2 NAc MSNs of mouse models as well as in neurons and non-neuronal cells of NAc in mouse models and depressed humans. We have considerable preliminary data to demonstrate the feasibility and likely success of these efforts. With the help of Project 4, we will also use a novel approach established by Project 3 investigators to map histone turnover in neurons and non-neuronal cells of this brain region from depressed humans and matched control subjects. Together, this work will provide new insight into the mechanisms controlling stress susceptibility vs. resilience over a lifetime.